Biochemical Studies on Vitamin B12 Part XI

In Part X, the authors published studies on vitamin B₁₂-like activity of cobalt-porphyrin-derivative prepared from radish-leaves, and the physiological significance of the compound as an active component in the intermediary metabolism of vitamin B₁₂ was described. , Afterward, pure chlorophyll a and b were isolated by column chromatography and the vitamin B₁₂-like activities of Co-porphyrin a and b were repeatedly confirmed.

Recently, effect of Co-porphyrin on the biosynthesis of real vitamin B₁₂ has been tested and the confirmation was also successfully completed by bioautography and paper electrophoresis.

In the present communication, it is discussed as of special importance that cobalt-porphyrin may be the active principle for the formation in vivo of vitamin B₁₂.     

Bile and detergent interaction with the radioassay for vitamin B12 binders using protein and dextran-covered charcoal

Bile acids and detergents such as Triton X-100, sodium dodecyl sulfate, and sodium deoxycholate interfered with the radioassay for vitamin B₁₂ binders. The effect was dependent on the concentration of the detergent. The detergents and bile acids were found, by use of [¹⁴C]glycocholic acid, to bind firmly to the covered charcoal in the assay, thereby blocking the binding of free vitamin B₁₂ to the charcoal. An increased ⁵⁷Co-labeled vitamin B₁₂ concentration was therefore observed in the final supernatant in the assay, giving rise to an apparent but false vitamin B₁₂-binding capacity.     

Influence of Vitamin B12 and Cocultures on the Growth of Dehalococcoides Isolates in Defined Medium

Bacteria belonging to the genus Dehalococcoides play a key role in the complete detoxification of chloroethenes as these organisms are the only microbes known to be capable of dechlorination beyond dichloroethenes to vinyl chloride (VC) and ethene. However, Dehalococcoides strains usually grow slowly with a doubling time of 1 to 2 days and have complex nutritional requirements. Here we describe the growth of Dehalococcoides ethenogenes 195 in a defined mineral salts medium, improved growth of strain 195 when the medium was amended with high concentrations of vitamin B₁₂, and a strategy for maintaining Dehalococcoides strains on lactate by growing them in consortia. Although strain 195 could grow in defined medium spiked with ~0.5 mM trichloroethene (TCE) and 0.001 mg/liter vitamin B₁₂, the TCE dechlorination and cellular growth rates doubled when the vitamin B₁₂ concentration was increased 25-fold to 0.025 mg/liter. In addition, the final ratios of ethene to VC increased when the higher vitamin concentration was used, which reflected the key role that cobalamin plays in dechlorination reactions. No further improvement in dechlorination or growth was observed when the vitamin B₁₂ concentration was increased to more than 0.025 mg/liter. In defined consortia containing strain 195 along with Desulfovibrio desulfuricans and/or Acetobacterium woodii and containing lactate as the electron donor, tetrachloroethene (~0.4 mM) was completely dechlorinated to VC and ethene and there was concomitant growth of Dehalococcoides cells. In the cultures that also contained D. desulfuricans and/or A. woodii, strain 195 cells grew to densities that were 1.5 times greater than the densities obtained when the isolate was grown alone. The ratio of ethene to VC was highest in the presence of A. woodii, an organism that generates cobalamin de novo during metabolism. These findings demonstrate that the growth of D. ethenogenes strain 195 in defined medium can be optimized by providing high concentrations of vitamin B₁₂ and that this strain can be grown to higher densities in cocultures with fermenters that convert lactate to generate the required hydrogen and acetate and that may enhance the availability of vitamin B₁₂.     

A comparison between different concentrations and sources of cobalt in goat kid nutrition

There have been extensive studies in sheep and cattle considering cobalt (Co) supplementation and its effects on vitamin B₁₂ concentrations in the body. However, there are limited studies on goats. The aim of this study was to compare two different sources of Co (sulfate v. glucoheptonate) at two different concentrations (0.25 and 0.5 mg/kg dry matter) in goat kid nutrition, and to evaluate the effects of these supplements on performance, serum vitamin B₁₂, blood biochemistry and rumen volatile fatty acids. For this purpose, 30 weaned male goat kids were randomly allotted to five treatments. Serum vitamin B₁₂ increased during the trial in the Co-supplemented groups. Co supplementation increased serum glucose concentrations. On day 35, Co-supplemented groups had greater glucose concentrations compared with control. Propionic+iso-butyric acid concentrations increased only in the 0.5 mg Co glucoheptonate treatment (P<0.05). Our results suggest that, despite the two sources of Co proving mostly similar, the main advantage of Co glucoheptonate compared with Co sulfate was in the ruminal synthesis of vitamin B₁₂. However, although providing Co at National Research Council recommendation levels maintained vitamin B₁₂ above or at normal concentrations, Co supplementation of the Co sufficient basal diet increased vitamin B₁₂ and glucose concentrations.     

STUDIES ON CHLOROPLAST DEVELOPMENT AND REPLICATION IN EUGLENA : I. Vitamin B12 and Chloroplast Replication

When Euglena gracilis is grown under vitamin B₁₂ deficiency conditions, the amount of protein and of chlorophyll per cell increase with decrease of B₁₂ in the medium and consequently in the cell. The increase in cell protein is proportional to and precedes an increase in the number of chloroplasts per cell. This replication of the chloroplasts under deficiency conditions is not accompanied by nuclear or cell division. It is concluded that chloroplast replication in Euglena gracilis is independent of nuclear and cellular replication, at least under B₁₂ deficiency conditions. We established a graph of the growth of Euglena under different concentrations of vitamin B₁₂ added to the growth medium, which permitted us to calculate that at least 22,000 molecules of vitamin B₁₂ per cell are required to give normal growth.     

The nature of the requirement of Saccharomyces carlsbergensis for vitamin B6

Saccharomyces carlsbergensis 4228, an organism widely used for determination of vitamin B₆, grows well without this vitamin if thiamine is also omitted from the basal medium, and an inoculum grown in a thiamine-low medium is used. Thiamine inhibits growth when added to such a medium. The thiazole moiety of thiamine, but not the pyrimidine, is also inhibitory, but less so than thiamine itself.Growth inhibition by thiamine is prevented by vitamin B₆. At low concentrations of thiamine, the amount of vitamin B₆ required for growth increases with the thiamine concentration; at concentrations of thiamine above 1 μg./10 ml. the vitamin B₆ requirement for growth remains essentially constant. Since these higher concentrations of thiamine have been used in methods that utilize this organism for determination of vitamin B₆ (1,2), the validity of these methods is confirmed.In the presence of thiamine, growth was also permitted by additions of the thiamine antagonist, neopyrithiamine. In this case, however, the relationship was fully competitive; i.e., the amount of neopyrithiamine required for growth increased regularly with the thiamine concentration. At concentrations considerably higher than those required for growth, neopyrithiamine again inhibited growth, and this inhibition was prevented by an increase in the thiamine concentration. Thus neopyrithiamine acts by lowering the effective thiamine concentration to subinhibitory levels; if excessive amounts are used, it prevents essential metabolic functions of thiamine and itself becomes toxic. The mechanism by which vitamin B₆ prevents thiamine toxicity is not known.The appearance of a requirement for certain growth factors because of inhibitory effects of other metabolically important compounds, rather than because of an intrinsic inability of the organism to synthesize the growth factor, may be much more common than the few recorded instances of this phenomenon indicate.     

Production of vitamin B12 by immobilized cells of a propionic acid bacterium

Summary The ability of immobilized cells of propionic acid bacteria to form vitamin B₁₂ has been investigated. Propionibacterium arl AKU 1251 having a considerable activity to produce the vitamin was selected as a test organism among six strains of propionic acid bacteria tested. The whole cells were entrapped with urethane prepolymers, photo-crosslinkable resin prepolymers or several other materials such as -carrageenan, agar or sodium alginate, and their vitamin B₁₂ productivity was compared. Based on the criteria of the convenience of preparation and the stability of the cell-entrapping gels, a hydrophilic urethane prepolymer, PU-9, was employed as gel material. Satisfactory vitamin B₁₂ production was obtained when 5–10 g of wet cells precultured to the late exponential growth phase were entrapped with 1 g of the prepolymer. Addition of a suitable amount of cobaltous ion and of 5,6-dimethyl benzimidazole to the culture medium was effective for the production of the vitamin by the immobilized cells. The repeated use of the immobilized cells was successfully achieved when a suitable amount of cells were entrapped and allowed the proliferation of cells inside gel matrices.     

EFFECT OF HORMONES AND VITAMIN B12 ON GAMETOPHORE DEVELOPMENT IN THE MOSS PYLAISIELLA SELWYNII

Bud formation in the moss Pylaisiella selwynii is greatly enhanced by cytokinins at concentrations as low as 10⁻¹²m , yet these buds usually fail to develop into normal gametophores. Various ratios at different concentrations of the cytokinin N-6-γ,γ-dimethylallylaminopurine to indoleacetic acid failed to enhance bud initiation over that obtained with cytokinin alone or to permit normal gametophore development. Deletion of the cobaltous ions from the culture medium prevented the appearance of the few gametophores usually formed in the complete medium, but different amounts of cobaltous ion did not significantly enhance initiation of gametophore development. Bud initiation was enhanced 3- to 20-fold by vitamin B₁₂ at 10⁻⁵m or by B₁₂ coenzyme at 10⁻⁴m , and the time of appearance of these buds was advanced by 6–12 days compared to control plants. At these concentrations of the B₁₂ compounds the buds formed normal gametophores, but at 10⁻⁴m vitamin B₁₂ they grew into callus-like masses similar to those obtained with cytokinins. Although the effects of B₁₂ on bud initiation and development mimicked those of cytokinins, except in permitting normal development, no additive or synergistic effects were observed when they were tested together. It is suggested that B₁₂ may play a regulatory role in the control of gametophore initiation and development in mosses.     

Impact of Vitamin B12 on Formation of the Tetrachloroethene Reductive Dehalogenase in Desulfitobacterium hafniense Strain Y51

Corrinoids are essential cofactors of reductive dehalogenases in anaerobic bacteria. Microorganisms mediating reductive dechlorination as part of their energy metabolism are either capable of de novo corrinoid biosynthesis (e.g., Desulfitobacterium spp.) or dependent on exogenous vitamin B₁₂ (e.g., Dehalococcoides spp.). In this study, the impact of exogenous vitamin B₁₂ (cyanocobalamin) and of tetrachloroethene (PCE) on the synthesis and the subcellular localization of the reductive PCE dehalogenase was investigated in the Gram-positive Desulfitobacterium hafniense strain Y51, a bacterium able to synthesize corrinoids de novo. PCE-depleted cells grown for several subcultivation steps on fumarate as an alternative electron acceptor lost the tetrachloroethene-reductive dehalogenase (PceA) activity by the transposition of the pce gene cluster. In the absence of vitamin B₁₂, a gradual decrease of the PceA activity and protein amount was observed; after 5 subcultivation steps with 10% inoculum, more than 90% of the enzyme activity and of the PceA protein was lost. In the presence of vitamin B₁₂, a significant delay in the decrease of the PceA activity with an ∼90% loss after 20 subcultivation steps was observed. This corresponded to the decrease in the pceA gene level, indicating that exogenous vitamin B₁₂ hampered the transposition of the pce gene cluster. In the absence or presence of exogenous vitamin B₁₂, the intracellular corrinoid level decreased in fumarate-grown cells and the PceA precursor formed catalytically inactive, corrinoid-free multiprotein aggregates. The data indicate that exogenous vitamin B₁₂ is not incorporated into the PceA precursor, even though it affects the transposition of the pce gene cluster.     

Influence of Zn<Superscript>2+</Superscript>, Co<Superscript>2+</Superscript> and dimethylbenzimidazole on vitamin B<Subscript>12</Subscript> biosynthesis by <Emphasis Type="Italic">Pseudomonas denitrificans</Emphasis>

Previous research has confirmed that cobalt ion and dimethylbenzimidazole (DMBI) are the precursors of vitamin B₁₂ biosynthesis, and porphobilinogen synthase (PBG synthase) is a zinc-requiring enzyme. In this paper, the effects of Zn²⁺, Co²⁺ and DMBI on vitamin B₁₂ production by Pseudomonas denitrificans in shake flasks were studied. Present experimental results demonstrated that the addition of the above mentioned three components to the fermentation medium could significantly stimulate the biosynthesis of vitamin B₁₂. The concentrations of zinc sulphate, cobaltous chloride and DMBI in the fermentation medium were further optimized with rotatable orthogonal central composite design and statistical analysis by Data Processing System (DPS) software. As a result, vitamin B₁₂ production was increased from 69.36 ± 0.66 to 78.23 ± 0.92 μg/ml.     

Vitamin B(12) in photosynthetic bacteria and methionine synthesis by Rhodopseudomonas spheroides

1. Assay of some photosynthetic bacteria for vitamin B₁₂ showed them to be relatively rich in this factor. Rhodopseudomonas spheroides, grown photosynthetically in Co²⁺-supplemented medium, contained about 100μg./g. dry wt. 2. Extracts of wild-type Rps. spheroides methylated homocysteine by a mechanism similar to the cobalamin-dependent pathway present in Escherichia coli. However, no mechanism similar to the cobalamin-independent N⁵-methyltetrahydrofolate–homocysteine transmethylase of E. coli could be detected in Rps. spheroides. 3. N⁵N¹⁰-Methylenetetrahydrofolate-reductase activity was found in Rps. spheroides. 4. A methionine-requiring mutant strain of Rps. spheroides (strain 2/33), which does not respond to homocysteine, made the same amount of vitamin B₁₂ as the parent organism. Extracts did not form methionine from N⁵-methyltetrahydrofolate and homocysteine even in the presence of cofactors shown to be necessary with the parent strain, and it is concluded that the mutant is blocked in the formation of the apoenzyme of a homocysteine-methylating system similar to the vitamin B₁₂-dependent one in E. coli.     

Characterization of vitamin B12 compounds in the fruiting bodies of shiitake mushroom (Lentinula edodes) and bed logs after fruiting of the mushroom

This study determined the vitamin B₁₂ content in commercially available dried fruiting bodies of shiitake mushroom, Lentinula edodes. The vitamin B₁₂ contents in dried donko-type fruiting bodies with closed caps (5.61 ± 3.90 μg/100 g dry weight), did not significantly differ from those of dried koushin-type fruiting bodies with open caps (4.23 ± 2.42 μg/100 g dry weight). The bed logs after fruiting of the mushroom also contained the vitamin B₁₂ levels similar to that in the dried shiitake fruiting bodies. To determine whether the dried shiitake fruiting bodies and their bed logs contained vitamin B₁₂ or other corrinoid compounds that are inactive in humans, we purified corrinoid compounds using an immunoaffinity column and identified vitamin B₁₂ using vitamin B₁₂-dependent Escherichia coli 215 bioautograms and liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) chromatograms. Dried shiitake fruiting bodies rarely contained an unnatural corrinoid vitamin B₁₂[c-lactone] that is inactive in humans. Given that shiitake mushroom lacks the ability to synthesize vitamin B₁₂ de novo, the vitamin B₁₂ found in dried shiitake fruiting bodies must have been derived from the bed logs.     

Common variant in FUT2 gene is associated with levels of vitamin B12 in Indian population

Vitamin B₁₂ is an essential micronutrient synthesized by microorganisms. Mammals including humans have evolved ways for transport and absorption of this vitamin. Deficiency of vitamin B₁₂ (either due to low intake or polymorphism in genes involved in absorption and intracellular transport of this vitamin) has been associated with various complex diseases. Genome-wide association studies have recently identified several common single nucleotide polymorphisms (SNPs) in fucosyl transferase 2 gene (FUT2) to be associated with levels of vitamin B₁₂—the strongest association was with a non-synonymous SNP rs602662 in this gene. In the present study, we attempted to replicate the association of this SNP (rs602662) in an Indian population since a significant proportion has been reported to have low levels of vitamin B₁₂ in this population. A total of 1146 individuals were genotyped for this SNP using a single base extension method and association with levels of vitamin B₁₂ was assessed in these individuals. Regression analysis was performed to analyze the association considering various confounding factors like for age, sex, diet, hypertension, diabetes mellitus and coronary artery disease status. We found that the SNP rs602662 was significantly associated with the levels of vitamin B₁₂ (p value < 0.0001). We also found that individuals adhering to a vegetarian diet with GG (homozygous major genotype) have significantly lower levels of vitamin B₁₂ in these individuals. Thus, our study reveals that vegetarian diet along with polymorphism in the FUT2 gene may contribute significantly to the high prevalence of vitamin B₁₂ deficiency in India.     

Effect of water soluble vitamins and their analogues on growth of Candida albicans II. Vitamin B12, thiamine,oxythiamine, neopyrithiamine,substituted pyrimidines and thiazoles

Summary By employing wide ranges in vitamin concentrations in biotin basal mineral synthetic medium, it was demonstrated that vitamin B₁₂ markedly stimulated the growth ofCandida albicans, the organism showing a partial dependency upon this vitamin. Growth inhibition by 5-fluorouracil was reversed non-competitively by vitamin B₁₂, suggesting that B₁₂ has a role in nucleic acid biosynthesis of the organism. Thiamine was growth stimulatory, the organism being partially dependent upon this vitamin as well. Neopyrithiamine and oxythiamine were growth inhibitory in thiamine-free biotin basal mineral synthetic medium although the halves of each inhibitor compound were non-inhibitory. Neopyrithiamine inhibition was reversed by intact thiamine but not by pyrimidine thiamine or thiazole thiamine; while oxythiamine inhibition was reversed by thiamine and pyrimidine thiamine but not by thiazole thiamine, the inference being drawn that oxythiamine selectively blocks utilization of pyrimidine thiamine. Twenty-seven different substituted pyrimidines, thiazoles and related thiamine compounds were all utilizable byC. albicans in thiamine-free basal synthetic mineral medium, the organism presumably synthesizing thiamine when presented with the constituent parts of these thiamine analogues. Substitution of sulfur of the thiazole ring with oxygen, as in -methyloxazolium, failed to produce an inhibitory compound forC. albicans. Acetylthiamine, allithiamine, cocarboxylase, tetrahydrothiamine and dihydrothiamine were equally as growth stimulatory as thiamine.     

Enrichment of some B-vitamins in plants with application of organic fertilizers

A review of the literature showed that plants grown with organic fertilizers often contain higher concentrations of vitamins B₁ (thiamin) and B₁₂ (cyanocobalamin) as compared with plants grown with inorganic fertilizers. Since plant roots were recently shown to be able to absorb B₁ and B₁₂, it was thus suspected that organic fertilizers (such as manure of diverse sources or sewage sludges which often contain relatively high concentrations of several vitamins) introduce additional vitamins into the soil which in turn leads to increased vitamins in the plants. This possibility was studied by measuring the B₁₂ content in the seeds of soybean and barley and in the leaves of spinach plants grown in soils amended with pure B₁₂ or cow dung (which is naturally rich in B₁₂). The addition of pure B₁₂ or cow dung did not alter the B₁₂ content in the soybean seeds but significantly increased that in the barley kernels and in the spinach leaves. For example, the addition of cow dung at the rate of 10 g kg^–1 increased the B₁₂ content in barley kernels by more than threefold (from 2.6 to 9.1 ng g^–1 DW) and in spinach leaves by close to twofold (from 6.9 to 17.8 ng g^–1 DW). Long-term addition of organic fertilizers to the soil also significantly increased the soil content of this vitamin. Since plants cannot synthesize B₁₂ and thus plant foods are normally fully devoid of (or have very low concentrations of) this vitamin, the finding that plants grown with organic fertilizers may contain relatively higher concentrations of this vitamin may have nutritional consequences in that the consumption of these plants by humans would inadvertently increase their intake of this vitamin. This may be of special benefit to people living by choice or by necessity on strict vegetarian diets who are known to be in danger of B₁₂ deficiency.     

Megaloblastic Anemia Associated with Surgically Produced Gastrointestinal Abnormalities

Two of the mechanisms for vitamin B₁₂ deficiency, leading to megaloblastic anemia, are the result of surgically produced abnormalities of the gastrointestinal tract. The basic mechanism is different for each lesion.Total gastrectomy results in complete lack of intrinsic factor which is necessary for vitamin B₁₂ absorption. It is believed that if patients survive long enough and are not given prophylactic vitamin B₁₂ therapy, all would develop megaloblastic anemia.Intestinal anastomosis leading to stasis of intestinal contents, with overgrowth of bacteria may cause vitamin B₁₂ deficiency through bacterial interference with the utilization of vitamin B₁₂.Use of radioactive vitamin B₁₂ (cobalt⁶⁰-labeled B₁₂) has led to a better understanding of the pathogenesis of both types of megaloblastic anemia. The radioactive vitamin provides a useful tool for study of its absorption from the gastrointestinal tract.     

UNEXPECTED RESPONSE TO VITAMIN B12 OF DOMINANT CENTRIC DIATOMS FROM THE SPRING BLOOM IN THE GULF OF MAINE (NORTHEAST ATLANTIC OCEAN)1,2

Twelve clones (seven species) representative of centric diatoms dominant in the spring phytoplankton bloom in the Gulf of Maine were isolated and rendered axenic. Genera included were Thalassiosira, Porosira and Chaetoceros. Unlike most centric diatoms studied previously, none of these has an absolute requirement for vitamin B_12. However, B₁₂ (5 ng.l^-1) stimulated growth of most clones by eliminating or reducing the lag phase and increasing the growth rate. Bloom population densities developed 4–54 days earlier with B₁₂ present. Several clones grown with B₁₂ removed more than 80% of the vitamin from the medium. When grown in vitamin-free medium the cells put 0.01–0.7 ng.l^-1 B₁₂ into the medium. We conclude that vitamin B₁₂ is of ecological significance even though the requirement for it is not absolute.     

Isolation of a novel Pseudomonas species SP2 producing vitamin B12 under aerobic condition

Vitamin B₁₂ is a complex biomolecule that acts as a cofactor for a variety of enzymes in microbial metabolism. Pseudomonas denitrificans is exclusively used as an industrial strain for the production of vitamin B₁₂ under aerobic conditions. However, only a few strains of Pseudomonas have been reported to possess the capability of producing this vitamin and they are strongly patent-protected. To improve the applicability of the vitamin B₁₂-producing microorganisms, a new isolate was obtained from municipal waste samples and characterized for its biological properties. The new isolate, designated as SP2, was identified to be a Pseudomonas species based on the sequence homology of its 16S rDNA. Pseudomonas species SP2 had essential genes for vitamin B₁₂ synthesis such as cobB and cobQ and produced a similar amount of vitamin B₁₂ (10.6 ± 0.05 μg/mL) as P. denitrificans ATCC 13867 in 24 h flask culture. SP2 grew well under aerobic condition with the maximum specific growth rate (µ _max ) of 0.91 ± 0.03/h, but showed a poor growth under micro-aerobic conditions. SP2 was resistant to antibiotics like streptomycin, carbenicillin, ampicillin, cefpodoxime, colistin, nalidixic acid and sparfloxacin. The ability of SP2 to grow faster and produce vitamin B₁₂ under aerobic conditions makes it a promising host for the production of some biochemicals requiring a coenzyme B₁₂-dependent enzyme, such as glycerol dehydratase.